DAG3 QC report
DAG3 Pilot (200 samples), QC report part II - bioinformatics QC vs Lab QC
- By: Ranko Gacesa, 05/10/2017
- FastQC was performed on 200 raw reads received from Novogene
- Results were linked to taxonomy reports (Arnau)
- Results were also compared to lab reports from UMCG and Novogene to identify potential issues
Identification of ‘problematic’ samples
How to define a ‘bad’ (metagenomic) sequencing run?
- Low DNA concentration?
- DNA contamination?
- ‘Bad’ result of diagnostic gel run?
- Low number of reads?
- High level of read duplication?
- Unusually low or high GC content?
- Low microbiome diversity?
- …
For purposes of this analysis, ‘problematic’ samples were defined as:
- samples with high sequence duplication (>=30% of total reads are duplicate)
- and/or GC content considerably different from rest of samples (<= 47% or >= 57.5%)
| sample | tot.seq | pct.gc | pct.dup | UMCG.C_ng_ul | novo.C_ng_ul | novo.Conclusion2 | tot.seq.corr |
|---|---|---|---|---|---|---|---|
| 7020000655314 | 13607727 | 57 | 42.3 | 41.6 | 9.6 | Regular prep possible | 7857782 |
| 7020000717918 | 14165712 | 46 | 40.9 | 32.0 | 7.4 | resend recommended | 8366270 |
| 7020000774217 | 24707796 | 46 | 27.9 | 58.1 | 4.0 | resend recommended | 17821733 |
| 7020000854013 | 13689929 | 48 | 32.6 | 34.8 | 6.6 | require low DNA library prep | 9229750 |
| 7020000866417 | 17151076 | 45 | 27.9 | 118.7 | 26.2 | Regular prep possible | 12373644 |
| 7020000871032 | 16918081 | 54 | 31.8 | 67.3 | 21.0 | Regular prep possible | 11530518 |
| 7020000876431 | 14700833 | 46 | 32.1 | 39.2 | 6.4 | resend recommended | 9985541 |
| 7020001036428 | 12699984 | 40 | 61.0 | 73.2 | 15.5 | Regular prep possible | 4953629 |
| 7020001093925 | 14347902 | 59 | 16.1 | 43.3 | 4.3 | resend recommended | 12034303 |
| 7020001128136 | 17080328 | 59 | 31.2 | 36.4 | 8.3 | require low DNA library prep | 11758098 |
LAB QC (Novogene)
- Sanity checks of Novogene QC follows:
LAB QC (Novogene)
- what they think they got from us:
LAB QC (UMCG)
Our DNA concentration
LAB QC (UMCG)
Batch effects testing (lab parameters):
UMCG: Box 24 seems to be problematic (all samples were labelled as Failed or Hold by Novogene)
- Box 24: extraction volume was 200 ul, rest of boxes were done with 100 ul
- different protocol?
Batch effects testing (FastQC results):
Batch effects testing (Taxonomy results):
UMCG QC (Nanodrop) vs Novogene QC (Qubit2):
- comparison of measurements at UMCG to Novogene report
Post-sequencing QC (FastQC)
FastQC parameters for Novogene QC groups:
FastQC parameters VS DNA concentration (UMCG Nanodrop)
FastQC parameters VS DNA concentration (Novogene Qubit2)
FastQC plots (different FastQC parameters)
Taxonomy
- QC results were related to taxonomy mapping of samples (Arnau’s data)
Taxonomy: Taxonomy (Species & Diversity) VS FastQC measurements
Taxonomy: Taxonomy (Species & Diversity) VS Lab measurements
260/280 measurements
